HIF-1α和hTERT shRNA双基因抑制脑胶质瘤生长的实验研究

The study of inhibitory effect of HIF-1α shRNA and hTERT shRNA on growth of human glioma

目的研究靶向缺氧诱导因子-1α（HIF-1α）和人端粒酶逆转录酶（hTERT）的短发夹RNA（shRNA），对裸鼠皮下人脑胶质瘤中HIF-1α、hTERT基因表达的抑制作用，及其对肿瘤增殖和细胞凋亡的影响。方法体外培养人脑胶质瘤U251细胞；建立人脑胶质瘤裸鼠皮下接种模型；将HIF-1α shRNA、hTERT、shRNA质粒转染入移植瘤中，观察肿瘤生长情况；HE染色法观察肿瘤组织的病理改变；Western blot检测HIF-1α、hTERT蛋白表达；Annexin V＋PI双染流式细胞仪检测凋亡情况。结果成功建立稳定的裸鼠U251皮下移植瘤模型；治疗5周后HIF-1α shRNA、hTERT shRNA组双基因干扰组肿瘤体积较单基因干扰组及对照组明显减小，抑瘤率为84．2％；病理结果显示双基因干扰组肿瘤生长受到抑制，微血管散在稀疏，大量肿瘤细胞坏死及凋亡；Western blot显示治疗组相应蛋白表达受抑制，双基因干扰组影响二者表达；流式细胞仪检测双基因干扰组肿瘤细胞凋亡率明显高于其他组（P〈0.01）。结论HIF-1α shRNA和hTERT shRNA均可在裸鼠移植瘤体内抑制相应蛋白表达及胶质瘤增殖生长，促进细胞凋亡，而双基因干扰作用更强。

Objective To explore the effect of silencing Hypoxia-induced factor-1α （ HIF-1α） gene and human telomerase reverse transcriptase（hTERT） gene by short hairpin RNAs （shRNAs） on growth and apoptosis of human gliomas in nude mice. Methods To culture human glioma U251 cells in vitro and establish models of Human glioma subcutaneous xenograft in nude mice. The volumes of tumors and the inhibition rates were measured five weeks after therapy. Xenograft tumors were observed with HE staining after plasmid therapy. Protein levels of HIF-1α and hTERT were detected by Western blot. Apoptosis of tumor cells staining with Annexin V and PI were measured by flow cytometer. Results The subcutaneous xenograft glioma model of nude mice were constructed steadily. The volumes of tumor in HIF-1α shRNA ＋ hTERT shRNA group was significantly decreased compared with blank plasmid group, PBS group and HIF- 1α/hTERT shRNA group, the inhibition rate was 84.2%. Rare natural tumor cells and blood vessel, massive necrosic and apoptotic tumor cells were observed. Protein levels of HIF-1α and/or hTERT were also inhibited. When treated with HIF-1α shRNA and hTERT shRNA, the two protein levels were also inhibited. The apoptosic rate of tumor cells in HIF-1α shRNA ＋ hTERT shRNA group was higher than other groups （ P 〈0. 01 ）. Conclusion HIF-1α shRNA or hTERT shRNA could inhibite the corresponding protein levels and inhibit the Multiplication and growth of gliomas, induce apoptosis of tumor cells. Moreover, the effects of HIF-1α shRNA with hTERT shRNA is better.